The ultimate goal of this proposed work will be to develop specific and potent, specific inhibitors of the enzyme Gamma-Glutamyl (gamma-GH). The rationale for this objective is based on evidence that has attributed resistance to the anti-folate drug Methotrexate (MTX) by certain tumor cells to increased cellular and extracellular levels of the enzyme. Therefore, the development of specific inhibitors of gamma-GH is expected to be therapeutically invaluable for combating MTX-resistant tumors by countering their mode of resistance. To effectively design specific inhibitors of gamma-GH, the structural requirements for efficient binding to the enzyme by small molecules must be clarified in addition to establishing the structural motifs necessary for inhibition of the enzyme. To address the first point, the synthesis and valuation for various substrate probes for the gamma-GH will be undertaken and suitable binding structures will be selected. Based on two possible mechanisms of action for the enzyme, two general classes of phosphorus-containing inhibitors will be synthesized and evaluated for inhibitory potency against human and bacterial gamma-GH in order to gain insight into the requisite structural and chemical features for selective inhibition. The above described studies will be undertaken concurrently and the information gained from each will be ultimately incorporated into the design of specific potent inhibitors of gamma-GM. Such designed inhibitors will be subsequently synthesized and evaluated for inhibition of human gamma-GH. It is anticipated that these compounds will be extremely potent inhibitors of gamma-GM and thus may provide hope for those patients suffering from a form of cancer that has acquired resistance to useful chemotherapeutic agents such as MTX.